Alpha-Conotoxin EI is the only peptide known to bind with much higher affinity to the agonist site at the alphadelta subunit interface of the electric organ acetylcholine receptor (AChR) than to the other agonist site of the alphagamma interface. The objective of this research proj4ect is to determine which amino acids on the delta subunit and on EI interact to produce this unique selectivity. The three specific aims to achieve this are 1) to identify the EI high-affinity determinant residues on the delta chain by making deltagamma and gammadelta chimeras and delta and gamma point mutants and measuring the loss or gain of EI high affinity when these are co-expressed in cells along with the wild-type ACHR subunits, 2) to identify the high-affinity determinant residues on EI by making EI analogues which substitute an alanine in which non-cysteine position and observing how this affects peptide affinity for wild-type ACHR and 3) to test if the high-affinity determinant residue pairs identified in Aims 1 and 2 interact with each other by using double- mutant cycle analysis. Peptide affinity will be estimated from its competition with the AChR-specific radioligand, 125I-alpha- bungarotoxin. These studies will give a clearer understanding of the structure of this receptor and how alpha-conotoxins bind to it. This should lead to a better understanding of homologous human receptors, such as the muscle and neuronal AChRs, and their involvement in various neurological disease processes. Alpha-Conotoxins like EI show clinical promise as potential neuromuscular and neuronal blocking agents and as site-specific probes to aid in the development of such agents.